Antiviral Agents, Uses Thereof and Methods for Their Preparation

ABSTRACT

The present disclosure relates to the use of antiviral agents that are cladoniamides and derivatives thereof such as the compound Formula I, for example, for the treatment of viral infections such as those caused by coronaviruses and/or flaviviruses. The present disclosure also includes antiviral agents and methods for their preparation. (I)

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims the benefit of priority from co-pendingU.S. provisional application Nos. 63/015,027 filed on Apr. 24, 2020 and63/038,248 filed on Jun. 12, 2020, the contents of both of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to antiviral agents. For example, thepresent disclosure relates to cladoniamide compounds that are antiviralagents against viruses such as coronaviruses and/or flaviviruses as wellas methods for their preparation and their use.

BACKGROUND

The Flaviviridae family of viruses are positive, single-stranded,enveloped ribonucleic acid (RNA) viruses found primarily in ticks andmosquitoes, which can lead to human infections. For example, members ofthe genus Flavivirus include viruses, like dengue virus (DENV), WestNile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV), Japaneseencephalitis virus (JEV) and yellow fever virus (YFV), which areimportant human pathogens. Most human flavivirus infections areincidental, since the viruses are unable to replicate the virus to highenough titers in their human host. However, there are exceptions in thecase of DENV, WNV, ZIKV and YFV. For some flaviviruses, there arereadily available and effective vaccines (i.e. YFV and JEV). However,there is no vaccine for ZIKV and the DENV vaccine, Dengvaxia™, which isa tetravalent chimeric vaccine that splices structural genes of the fourdengue viruses onto a 17D yellow fever backbone, has proved to be lessthan optimal. Furthermore, because DENV, ZIKV, and WNV are all dual-hostmosquito-borne flaviviruses, co-infections are common. Currently, thereis no treatment for ZIKV and no specific medicine to treat DENV or WNV,so antiviral agents against flavivirus infections are needed urgently.

The Coronaviridae family of viruses are enveloped, positive-sense,single-stranded RNA viruses, which cause infectious diseases in mammalsand birds. In humans, coronaviruses often cause respiratory infections,ranging from only mild symptoms, such as in the common cold, to moreserious symptomatic courses such as in severe acute respiratory syndrome(SARS, first reported in 2002), Middle East respiratory syndrome (MERS,first reported in 2012), or coronavirus disease (COVID-19, firstreported in 2019), which for some patients can be lethal. Currently,there are no vaccines or antiviral drugs to prevent or treat most humancoronavirus infections. The alarming rate at which new coronaviruseshave been emerging and spreading over the past 20 years is a reason whythere exists an urgent need to find new antiviral agents useful againstcoronavirus infections.

SUMMARY

Cladoniamide A produced by actinomycete Streptomyces unicialis is activeagainst coronavirus and flavivirus. While S. unicialis also producesother cladoniamides, such as e.g. cladoniamide D, cladoniamide Dpossessed no activity against DENV and ZIKV. Similar results were alsofound for SARS-CoV-2. Cladoniamide E and cladoniamide F similarly showedno activity against ZIKV. Cladoniamide A was found to be a highlyeffective antiviral against the UK strain of SARS-CoV-2 (202012/01),with an ED₅₀ of about 800 pM. Cladoniamide A and cladoniamide C bothshowed anti-ZIKV activity in brain organoids infected with ZIKV.

Accordingly, the present disclosure includes a use of a compound of theFormula I or a pharmaceutically acceptable salt thereof for treatment ofa viral infection in a subject in need thereof:

wherein each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or one ormore of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a use of a compound of the FormulaI or a pharmaceutically acceptable salt thereof for preparation of amedicament for treatment of a viral infection in a subject in needthereof:

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

In an embodiment, the compound of Formula I is a compound of FormulaI(a):

wherein

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃; or

Z⁶ and Z⁷ together form an optionally substituted aromatic ring, and Z¹,Z², Z³, Z⁴, and Z⁵ are each independently H, halo, optionallysubstituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″,NR′COR″, OR′, SR′, CN, or CF₃;

R¹, R², R³, and R⁴ are each independently H or an optionally substitutedalkyl; and

R′ and R″ are each independently H, or an optionally substituted alkyl.

In an embodiment, R¹ is C₁₋₆alkyl. In another embodiment, R¹ is methyl.

In an embodiment, R² and R⁴ are both H.

In an embodiment, R³ is H.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is chloro.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring orunsubstituted aromatic ring. In another embodiment, Z⁶ and Z⁷ togetherform an unsubstituted, 6-membered aromatic ring.

In an embodiment, the compound is a compound of Formula III:

In an embodiment, the compound is cladoniamide A.

In an embodiment, the compound or the pharmaceutically acceptable saltthereof is the compound.

In an embodiment, the viral infection is caused by one or morecoronavirus and/or flavivirus. In another embodiment, the viralinfection is caused by one or more coronavirus. In a further embodiment,the viral infection is caused by one or more of severe acute respiratorysyndrome (SARS) coronavirus-1 (SARS-CoV-1), SARS coronavirus-2(SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus(MERS-CoV) and human coronavirus 229E (HCoV-229E). In anotherembodiment, the viral infection is caused by SARS-CoV-2. In anotherembodiment, the SARS-CoV-2 is SARS-CoV-2 South Africa (501Y.V2),SARS-CoV-2 UK (VOC 202012/01) or SARS-CoV-2 Nigeria. In anotherembodiment, the viral infection is caused by HCoV-229E. In anembodiment, the viral infection is caused by one or more flavivirus. Inanother embodiment, the viral infection is caused by one or more ofdengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV) and yellow fever virus(YFV). In another embodiment, the viral infection is caused by one ormore of West Nile virus (WNV), Powassan virus (POWV), Japaneseencephalitis virus (JEV) and yellow fever virus (YFV). In an embodiment,the viral infection is caused by dengue virus (DENV). In an embodiment,the viral infection is caused by Zika virus (ZIKV).

In an embodiment, the subject is a human.

In an embodiment, the compound is formulated for administration in apharmaceutical composition comprising the compound and optionally apharmaceutically acceptable carrier.

The present disclosure also includes a compound of Formula I(b):

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl;

or a pharmaceutically acceptable salt thereof,

with the proviso that said compound is not exactly:

or wherein R⁵ is C₁₋₄akyl, X² is selected from H and halo, and R¹ isselected from H, halo and OH.

The present disclosure also includes a use of a compound of Formula I ora pharmaceutically acceptable salt thereof for inhibitingvacuolar-H⁺ATPase (V-ATPase):

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

In an embodiment, the compound of Formula I is a compound of FormulaI(a):

wherein

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃; or

Z⁶ and Z⁷ together form an optionally substituted aromatic ring, and Z¹,Z², Z³, Z⁴, and Z⁵ are each independently H, halo, optionallysubstituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″,NR′COR″, OR′, SR′, CN, or CF₃;

R¹, R², R³, and R⁴ are each independently H or an optionally substitutedalkyl; and

R′ and R″ are each independently H, or an optionally substituted alkyl.

In an embodiment, R¹ is C₁₋₆alkyl. In another embodiment, R¹ is methyl.

In an embodiment, R² and R⁴ are both H.

In an embodiment, R³ is H.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is chloro.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring orunsubstituted aromatic ring. In another embodiment, Z⁶ and Z⁷ togetherform an unsubstituted, 6-membered aromatic ring.

In an embodiment, the compound is a compound of Formula III:

In an embodiment, the compound is cladoniamide A.

In an embodiment, the compound or the pharmaceutically acceptable saltthereof is the compound.

In an embodiment, the inhibition of V-ATPase is in a subject.

In an embodiment, the subject suffers from a viral infection.

In an embodiment, the viral infection is caused by one or morecoronavirus and/or flavivirus. In another embodiment, the viralinfection is caused by one or more coronavirus. In a further embodiment,the viral infection is caused by one or more of severe acute respiratorysyndrome (SARS) coronavirus-1 (SARS-CoV-1), SARS coronavirus-2(SARS-CoV-2), Middle East respiratory syndrome (MERS) coronavirus(MERS-CoV) and human coronavirus 229E (HCoV-229E). In anotherembodiment, the viral infection is caused by SARS-CoV-2. In anotherembodiment, the SARS-CoV-2 is SARS-CoV-2 South Africa (501Y.V2),SARS-CoV-2 UK (VOC 202012/01) or SARS-CoV-2 Nigeria. In anotherembodiment, the viral infection is caused by HCoV-229E. In anembodiment, the viral infection is caused by one or more flavivirus. Inanother embodiment, the viral infection is caused by one or more ofdengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV) and yellow fever virus(YFV). In another embodiment, the viral infection is caused by one ormore of West Nile virus (WNV), Powassan virus (POWV), Japaneseencephalitis virus (JEV) and yellow fever virus (YFV). In an embodiment,the viral infection is caused by dengue virus (DENV). In an embodiment,the viral infection is caused by Zika virus (ZIKV).

In an embodiment, the subject is a human.

In an embodiment, the compound is formulated for administration in apharmaceutical composition comprising the compound and optionally apharmaceutically acceptable carrier.

One embodiment of the present disclosure comprises the use of a compoundof Formula I(a) for treatment of a viral infection:

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, independently may be H, halogen,an alkyl, optionally substituted, an aromatic cycle, an aromaticheterocycle, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃; Z⁶ and Z⁷, optionally, may be forming an aromatic ring that,optionally, may be further substituted; and wherein R¹, R², R³, and R⁴,independently may be H or an, optionally substituted, alkyl; and R′ andR″ independently may be H, or an, optionally substituted, alkyl. Whereinsaid viral infection may be caused by one or more coronavirus and/orflavivirus. Wherein said viral infection may be caused by one or morecoronavirus. Wherein said coronavirus may be Severe Acute RespiratorySyndrome (SARS) coronavirus-1 (SARS-CoV-1), or coronavirus-2(SARS-CoV-2), or Middle East Respiratory Syndrome (MERS) coronavirus(MERS-CoV). Wherein said coronavirus may be SARS-CoV-2. Wherein saidcoronavirus may be human coronavirus 229E (HCoV-229E). Wherein saidviral infection may be caused by one or more flavivirus. Wherein saidviral infection may be mosquito-borne. Wherein said flavivirus may beDengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV), Yellow Fever virus(YFV), or a combination thereof.

One embodiment of the present disclosure comprises the use of a compoundof Formula II(a) for treatment of a viral infection:

wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, and X⁸ independently may be H,halogen, an alkyl, optionally substituted, an aromatic cycle, anaromatic heterocycle, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′,CN, or CF₃; R¹, R², R³, R⁴, and R⁵, independently may be H or an,optionally substituted, alkyl; and R′ and R″ independently may be H, oran, optionally substituted, alkyl. Wherein said viral infection may becaused by one or more coronavirus and/or flavivirus. Wherein said viralinfection may be caused by one or more coronavirus. Wherein saidcoronavirus may be Severe Acute Respiratory Syndrome (SARS)coronavirus-1 (SARS-CoV-1), or coronavirus-2 (SARS-CoV-2), or MiddleEast Respiratory Syndrome (MERS) coronavirus (MERS-CoV). Wherein saidcoronavirus may be SARS-CoV-2. Wherein said coronavirus may be humancoronavirus 229E (HCoV-229E). Wherein said viral infection may be causedby one or more flavivirus. Wherein said viral infection may bemosquito-borne. Wherein said flavivirus may be Dengue virus (DENV), WestNile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV), Japaneseencephalitis virus (JEV), Yellow Fever virus (YFV), or a combinationthereof.

A further embodiment the present disclosure comprises the use of acompound of Formula III for the treatment of a viral infection:

Wherein said viral infection may be caused by one or more coronavirusand/or flavivirus. Wherein said viral infection may be caused by one ormore coronavirus. Wherein said coronavirus may be Severe AcuteRespiratory Syndrome (SARS) coronavirus-1 (SARS-CoV-1), or coronavirus-2(SARS-CoV-2), or Middle East Respiratory Syndrome (MERS) coronavirus(MERS-CoV). Wherein said coronavirus may be SARS-CoV-2. Wherein saidcoronavirus may be human coronavirus 229E (HcoV-229E). Wherein saidviral infection may be caused by one or more flavivirus. Wherein saidviral infection may be mosquito-borne. Wherein said flavivirus may beDengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV), Yellow Fever virus(YFV), or a combination thereof. Wherein said viral infection may becaused by DENV or ZIKV, or a combination thereof.

One embodiment of the present disclosure comprises the use ofcladoniamide A for treatment of a flavivirus infection. Wherein saidcladoniamide A has a chemical structure of Formula III(a):

Wherein said flavivirus may be Dengue virus (DENV), West Nile virus(WNV), Zika virus (ZIKV), Powassan virus (POWV), Japanese encephalitisvirus (JEV), Yellow Fever virus (YFV), or a combination thereof. Whereinsaid flavivirus may be DENV or ZIKV, or a co-infection thereof. Whereinsaid flavivirus may be DENV-3. Wherein said flavivirus may be DENV-4.Wherein said flavivirus may be ZIKV.

Another embodiment of the present disclosure comprises the use ofcladoniamide A for treatment of a coronavirus infection. Wherein saidcladoniamide A has a chemical structure of Formula III(a):

Wherein said viral infection may be caused by one or more coronavirus.Wherein said coronavirus may be Severe Acute Respiratory Syndrome (SARS)coronavirus-1 (SARS-CoV-1), or coronavirus-2 (SARS-CoV-2), or MiddleEast Respiratory Syndrome (MERS) coronavirus (MERS-CoV). Wherein saidcoronavirus may be SARS-CoV-2. Wherein said coronavirus may be humancoronavirus 229E (HcoV-229E).

A further embodiment of the present disclosure comprises the use ofent-cladoniamide A for treatment of a flavivirus infection. Wherein saident-cladoniamide A has a chemical structure of Formula III (b):

Wherein said flavivirus may be Dengue virus (DENV), West Nile virus(WNV), Zika virus (ZIKV), Powassan virus (POWV), Japanese encephalitisvirus (JEV), Yellow Fever virus (YFV), or a combination thereof.

A further embodiment of the present disclosure comprises the use ofent-cladoniamide A for treatment of a coronavirus infection, whereinsaid ent-cladoniamide A has a chemical structure of Formula III(b):

Wherein said viral infection may be caused by one or more coronavirus.Wherein said coronavirus may be Severe Acute Respiratory Syndrome (SARS)coronavirus-1 (SARS-CoV-1), or coronavirus-2 (SARS-CoV-2), or MiddleEast Respiratory Syndrome (MERS) coronavirus (MERS-CoV). Wherein saidcoronavirus may be SARS-CoV-2. Wherein said coronavirus may be humancoronavirus 229E (HcoV-229E).

One embodiment of the present disclosure provides a method of use of acompound of Formula I(a) for inhibiting vacuolar-H⁺ATPase (V-ATPase):

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, independently may be H, halogen,an alkyl, optionally substituted, an aromatic cycle, an aromaticheterocycle, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃; Z⁶ and Z⁷, optionally, may be forming an aromatic ring that,optionally, may be further substituted; and wherein R¹, R², R³, and R⁴,independently may be H or an, optionally substituted, alkyl; and R′ andR″ independently may be H, or an, optionally substituted, alkyl, saidmethod comprising: providing a compound of Formula I(a) to a subject inneed of V-ATPase inhibition. Wherein said subject may be a human or ananimal. Wherein said subject may suffer from a viral infection. Whereinsaid viral infection may be a coronavirus infection. Wherein said viralinfection may be caused by one or more coronavirus. Wherein saidcoronavirus may be Severe Acute Respiratory Syndrome (SARS)coronavirus-1 (SARS-CoV-1), or coronavirus-2 (SARS-CoV-2), or MiddleEast Respiratory Syndrome (MERS) coronavirus (MERS-CoV). Wherein saidcoronavirus may be SARS-CoV-2. Wherein said coronavirus may be humancoronavirus 229E (HcoV-229E). Wherein said viral infection may be aflavivirus infection. Wherein said flavivirus may be Dengue virus(DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV),Japanese encephalitis virus (JEV), Yellow Fever virus (YFV), or acombination thereof. Wherein said viral infection may be DENV or ZIKV,or a combination thereof.

One embodiment of the present disclosure comprises a compound of FormulaI(a):

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, independently may be H, halogen,an alkyl, optionally substituted, an aromatic cycle, an aromaticheterocycle, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃; R¹, R², R³, and R⁴, independently may be H or an, optionallysubstituted, alkyl; and R′ and R″ independently may be H, or an,optionally substituted, alkyl.

Another embodiment of the present disclosure comprises a compound ofFormula II(b):

wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, and XV independently may be H,halogen, an alkyl, optionally substituted, an aromatic cycle, anaromatic heterocycle, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′,CN, or CF₃; R¹, R², R³, R⁴, and R⁵, independently may be H or an,optionally substituted, alkyl; and R′ and R″ independently may be H, oran, optionally substituted, alkyl with the proviso that said compound isnot exactly:

A further embodiment of the present disclosure provides a biosyntheticmethod for producing a compound of Formula I(a) comprising cultivating abacterium in a culture medium so to cause accumulation of said compoundof Formula I(a) in said culture medium, and optionally isolating saidcompound of Formula I(a) from said culture medium.

A further embodiment of the present disclosure provides a biosyntheticmethod for producing a compound of Formula II(a) comprising cultivatinga bacterium in a culture medium so to cause accumulation of saidcompound of Formula II(a) in said culture medium, and optionallyisolating said compound of Formula II(a) from said culture medium.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the disclosure, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should rather be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the disclosure will now be described in greaterdetail with reference to the attached drawings, in which:

FIG. 1 is a plot to determine cytotoxicity concentration 50% (CC₅₀) ofcladoniamide A (CA) in A549 cells. Error bars represent SD among twobiological replicates.

FIG. 2 is a plot to determine cytotoxicity concentration 50% (CC₅₀) ofCA in U87 cells. Error bars represent SD among two biologicalreplicates.

FIG. 3 is a plot showing that CA, but not cladoniamide D (CD), exhibitsnanomolar antiviral activity against ZIKV. Average viral titres from twoindependent experiments (A549 cells) or three independent experiments(U87 MG cells) are shown. Significance was measured by a 2-tailedunpaired Student's t-test: *, p<0.05; ****, p<0.0001.

FIG. 4 shows IC₅₀ and therapeutic index (TI) values of PF-429242 (PF)and CA against ZIKV infection from extracellular viral NS1 enzyme-linkedimmunosorbent assay (ELISA) results (upper and middle plots,respectively) and IC₅₀ and TI values of CA from plaque assay (PFU)results (lower plot). Error bars represent SD among 2 biologicalreplicates.

FIG. 5 shows that in contrast to NITD-008, CA is not a ZIKV replicationinhibitor. Error bars represent SD among 3 biological replicates.

FIG. 6 shows that CA is dual inhibitor of ZIKV infection targeting theentry and maturation steps. Error bars represent SEM among 3 biologicalreplicates. Significance was calculated by a 2-tailed unpaired Student'st-test. *, p<0.05.

FIG. 7 shows results for CA (upper right image), cladoniamide D (CD;middle left image), cladoniamide E (CE; middle right image); andcladoniamide F (lower left image) in comparison to a control (upper leftimage). Cultured A549 cells were treated with 15 μM of the cladoniamidebefore being washed with PBS and infected with ZIKV at MOI 1.0 for 48 h.Cells were labeled for viral dsRNA then imaged using the CellomicsArrayScan™ VTI.

FIG. 8 shows results for DENV-2 (upper plots), DENV-3 (middle plots) andDENV-4 (lower plots), showing CA is a potential broad-spectrumanti-flavivirus agent. Error bars represent SD among 3 biologicalreplicates. Cultured A549 cells were treated with the indicatedconcentrations of CA (1=Mock, 2=0 M, 3=1 μM, 4=5 M, 5=10 M, 6=20 M) orPF-429242 (7=15 M) for 1 h before being washed with PBS and infectedwith DENV-2, DENV-3, or DENV-4 at MOI 1.0 for 48 h. Cells were labeledfor viral dsRNA with Hoechst dye then imaged and quantified using theCellomics ArrayScan™ VTI High Content Screening system.

FIG. 9 shows results for DENV-2 (upper plots), DENV-3 (middle plots) andDENV-4 (lower plots), for Huh7.5.1 cells (left plots) and U87 cells(right plots) showing CA is a potential broad-spectrum anti-flavivirusagent. Cultured Huh7.5.1 cells (left plots) or U87 cells (right plots)were treated with the indicated concentrations of CA (1=Mock, 2=0 M, 3=1M, 4=5 M, 5=10 M, 6=20 M) or PF-429242 (7=15 M) for 1 h before beingwashed with PBS and infected with DENV-2, DENV-3, or DENV-4 at MOI 1.0for 48 h. Cells were labeled for viral dsRNA with Hoechst dye thenimaged and quantified using the Cellomics ArrayScan™ VTI.

FIG. 10 shows molecular modelling of hVma3.

FIG. 11 shows fluorescence microscopy images of cultured A549 cellswithout treatment (upper left), and treated with Bafilomycin A1 (100 nM;upper right), CA (250 nM; lower left) and CD (1000 nM; lower right) for6 hours then treated with acridine orange. CA but not CD inhibited vitalstaining with acridine orange of the intracellular organelles of theA549 cells.

FIG. 12 shows additional fluorescence microscopy images of cultured A549cells without treatment (upper left and right), and treated with CA (250nM; lower left and right) for 6 hours then treated with acridine orange,showing CA activity as a V-ATPase inhibitor.

FIG. 13 shows fluorescence microscopy images of cultured A549 cellswithout treatment (top row), and treated with Bafilomycin A1 (100 nM;second row from top), CA (250 nM; second row from bottom) and CD (250nM; bottom row) for the indicated time periods then treated withacridine orange, showing the time course of CA-mediated inhibition.

FIG. 14 shows the results of studies of cladoniamide A against humancoronavirus 229E (HCoV-229E) in hepatoma cells (Huh 7.5 cells). AnIF-based assay was used to monitor viral dsRNA in infected cells. CAinhibits HCoV-229E infections in human cells, acting as an indirectantiviral agent targeting human V-ATPase, EC₅₀=15 nM.

FIG. 15 shows plots of CA activity against SARS-CoV-2 in comparison toCD which showed no antiviral effect based on intracellular viral RNA(upper plot) and intracellular SARS-CoV-2 nucleocapsid protein (lowerplot) in Human Caco2 cells pre-treated with the indicated amounts of CAor CD before adding the virus.

FIG. 16 is a plot of CA activity against the indicated SARS-CoV-2variants based on intracellular SARS-CoV-2 nucleocapsid protein in humanCalu-3 cells pre-treated with the indicated amounts of CA before addingthe virus (n=1).

FIG. 17 is a schematic showing a general method used in the examples ofthe present disclosure to study treatment of brain organoids with CA orcladoniamide C (CC).

FIG. 18 is a plot showing extracellular ZIKV titer for four brainorganoid systems after treatment with 1 μM CA for 72 hours in comparisonto untreated controls.

FIG. 19 is a plot showing toxicity results for brain organoids treatedwith 1 μM CA.

FIG. 20 shows plots of NS1 results for individual technical replicates(upper) and averaged values (lower) of brain organoids treated with 10μM CA or CC for 69 hours.

FIG. 21 shows plots of dsRNA results for individual technical replicates(upper) and averaged values (lower) of brain organoids treated with 10μM CA or CC for 69 hours.

DETAILED DESCRIPTION I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the disclosure herein described for which they would beunderstood to be suitable by a person skilled in the art.

Terms of degree such as “about” and “approximately” as used herein meana reasonable amount of deviation of the modified term such that the endresult is not significantly changed. These terms of degree should beconstrued as including a deviation of at least ±5% of the modified termif this deviation would not negate the meaning of the term it modifies.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items ispresent or used.

Numeric ranges are inclusive of the numbers defining the range.

As used in this disclosure, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.

As used herein, the words “comprising” (and any form thereof, such as“comprise” and “comprises”), “having” (and any form thereof, such as“have” and “has”), “including” (and any form thereof, such as “include”and “includes”) or “containing” (and any form thereof, such as “contain”and “contains”), are inclusive or open-ended and do not excludeadditional, unrecited elements or process/method steps. As used herein,the word “consisting” and its derivatives are intended to be close-endedterms that specify the presence of the stated features, elements,components, groups, integers and/or steps, and also exclude the presenceof other unstated features, elements, components, groups, integersand/or steps. The term “consisting essentially of”, as used herein, isintended to specify the presence of the stated features, elements,components, groups, integers, and/or steps as well as those that do notmaterially affect the basic and novel characteristic(s) of thesefeatures, elements, components, groups, integers and/or steps.

The term “suitable” as used herein means that the selection of theparticular compound and/or conditions would depend on the specificsynthetic manipulation to be performed, and/or the identity of thecompound(s) to be transformed, but the selection would be well withinthe skill of a person skilled in the art. All method steps describedherein are to be conducted under conditions sufficient to provide theproduct shown. A person skilled in the art would understand that allreaction conditions, including, for example, reaction solvent or lackthereof, reaction time, reaction temperature, reaction pressure,reactant ratio and whether or not the reaction should be performed underan anhydrous or inert atmosphere, can be varied to optimize the yield ofthe desired product and it is within their skill to do so.

The term “halo” as used herein refers to a halogen atom and includes F,Cl, Br and I. In an embodiment of the present disclosure, halo ischloro.

The term “alkyl” as used herein, whether it is used alone or as part ofanother group, means straight or branched chain, saturated alkyl groups.The number of carbon atoms that are possible in the referenced alkylgroup are indicated by the numerical prefix “C_(n1-n2)”. For example,the term C₁₋₆alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbonatoms.

The term “cycloalkyl” as used herein, whether it is used alone or aspart of another group, means a mono- or bicyclic, saturated cycloalkylgroup. The number of carbon atoms that are possible in the referencedcycloalkyl group are indicated by the numerical prefix “C_(n1-n2)”. Forexample, the term C₃₋₁₀cycloalkyl means a cycloalkyl group having 3, 4,5, 6, 7, 8, 9 or 10 carbon atoms. When a cycloalkyl group contains morethan one cyclic structure or rings, the cyclic structures may be fused,bridged, spiro connected or linked by a single bond. The term “fused” asused herein in reference to a first cyclic structure being “fused” witha second cyclic structure means the first cyclic structure and thesecond cyclic structure share at least two adjacent atoms therebetween.The term “bridged” as used herein in reference to a first cyclicstructure being “bridged” with a second cyclic structure means the firstcyclic structure and the second cyclic structure share at least twonon-adjacent atoms therebetween. The term “spiro connected” in referenceto a first cyclic structure being “spiro connected” with a second cyclicstructure means the first cyclic structure and the second cyclicstructure share one atom therebetween.

The term “alkylene” as used herein, whether it is used alone or as partof another group, means a straight or branched chain, bivalent form ofan alkane, that is, a saturated carbon chain that links two othergroups. The number of carbon atoms that are possible in the referencedalkylene group are indicated by the numerical prefix “C_(n1-n2)”. Forexample, the term C₁₋₆alkylene means an alkylene group having 1, 2, 3,4, 5 or 6 carbon atoms.

The terms “aryl” and “aromatic cycle” as used herein, whether used aloneor as part of another group, refers to groups that contain at least onearomatic ring. When an aryl group contains more than one aromatic ringthe terms “aryl” and “aromatic cycle” as used herein include condensedaromatic systems. In an embodiment, the aryl group contains from 6, 9,10 or 14 atoms, such as phenyl, naphthyl, indanyl or anthracenyl. Thenumber of carbon atoms that are possible in the referenced aryl groupare indicated by the numerical prefix “C_(n1-n2)”. For example, the termC₆₋₁₀aryl means an aryl group having 6, 7, 8, 9 or 10 carbon atoms.

The terms “heteroaryl” and “aromatic heterocycle” as used herein,whether used alone or as part of another group, refers to an aromatic,ring-containing group having one or more multivalent heteroatoms (forexample, heteroatoms independently selected from N, O and S), as a partof the ring structure. In an embodiment of the present disclosure, theheteroaryl includes at least 5 and up to 20 atoms in the ring(s).Heteroaryl groups may contain more than one ring.

The term “carbocyclic” as used herein in reference to a “carbocyclicring”, refers to a group containing at least one ring in which all ofthe atoms of the ring(s) are carbon atoms.

The term “heterocyclic” as used herein in reference to a “heterocyclicring”, refers to a group containing at least one ring, in which at leastone ring in the group has one or more multivalent heteroatoms (forexample, heteroatoms independently selected from N, O and S), as a partof the ring structure. In an embodiment of the present disclosure, theheterocyclic ring includes at least 5 and up to 20 atoms in the ring(s).

The term “haloalkyl” as used herein refers to an alkyl group wherein oneor more, including all of the available hydrogen atoms are replaced by ahalogen atom. The number of carbon atoms that are possible in thereferenced haloalkyl group are indicated by the numerical prefix“C_(n1-n2)”. For example, the term C₁₋₆haloalkyl means a haloalkyl grouphaving 1, 2, 3, 4, 5 or 6 carbon atoms. In an embodiment, the halogen isa fluorine, in which case the haloalkyl is optionally referred to hereinas a “fluoroalkyl” group. It is an embodiment that all of the hydrogenatoms are replaced by fluorine atoms.

The term “substituted” as used herein in reference to a group refers tosuch a group wherein one or more, including all of the availablehydrogen atoms are replaced by a substituent. In an embodiment, thesubstituents are selected from H, halo, alkyl, cycloalkyl,alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′,CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′,SO₂NR′R″, CN or haloalkyl, wherein R′ and R″ are each independently H,alkyl or alkylene-aryl.

The term “cladoniamide A” as used herein refers to a compound with anIUPAC name computed by LexiChem 2.6.6 of“(11S,15R)-7-chloro-11,15-dihydroxy-23-methoxy-13-methyl-3,13,16-triazahexacyclo[14.7.0.0^(2,10).0^(4,9).0^(11,15).0^(17,22)]tricosa-1(23),2(10),4(9),5,7,17,19,21-octaene-12,14-dione”and having the following chemical structure:

The term “cladoniamide C” as used herein refers to a compound with anIUPAC name computed by LexiChem 2.6.6 of“(11S,15R)-11,15-dihydroxy-23-methoxy-13-methyl-3,13,16-triazahexacyclo[14.7.0.0^(2,10).0^(4,9).0^(11,15).0^(17,22)]tricosa-1(23),2(10),4,6,8,17,19,21-octaene-12,14-dione”and having the following chemical structure:

The term “cladoniamide D” as used herein refers to a compound with anIUPAC name computed by LexiChem 2.6.6 of“(12R)-7-chloro-12-hydroxy-20-methoxy-N-methyl-11-oxo-3,13-diazapentacyclo[11.7.0.0^(2,10).0^(4,9).0^(14,19)]icosa-1(20),2(10),4(9),5,7,14,16,18-octaene-12-carboxamide”and having the following chemical structure:

The term “cladoniamide E” as used herein refers to a compound with anIUPAC name computed by LexiChem 2.6.6 of“(12R)-7,17-dichloro-12-hydroxy-20-methoxy-N-methyl-11-oxo-3,13-diazapentacyclo[11.7.0.0^(2,10).0^(4,9).0^(14,19)]icosa-1(20),2(10),4(9),5,7,14(19),15,17-octaene-12-carboxamide”and having the following chemical structure:

The term “cladoniamide F” as used herein refers to a compound with anIUPAC name computed by LexiChem 2.6.6 of“(11R)-7-chloro-11-hydroxy-20-methoxy-N-methyl-12-oxo-3,13-diazapentacyclo[11.7.0.0^(2,10).0^(4,9).0^(14,19)]icosa-1(20),2(10),4(9),5,7,14,16,18-octaene-11-carboxamide”and having the following chemical structure:

In embodiments of the disclosure, the compounds described herein have atleast one asymmetric center. Where compounds possess more than oneasymmetric center, they may exist as diastereomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present disclosure. It is to befurther understood that while the stereochemistry of the compounds maybe as shown in any given compound listed herein, such compounds may alsocontain certain amounts (e.g. less than 20%, optionally less than 10%,optionally less than 5%, optionally less than 3%) of the correspondingcompound having alternate stereochemistry.

The term “subject” as used herein includes all members of the animalkingdom including mammals and birds (class Aves), and optionally refersto humans.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects, for example, mammals (such as humans) or birds.

The term “pharmaceutically acceptable salt” as used herein means an acidaddition salt or a base addition salt that is compatible with thetreatment of subjects.

An “acid addition salt that is compatible with the treatment ofsubjects” is any non-toxic inorganic or organic salt of any basiccompound. Basic compounds that form an acid addition salt include, forexample, compounds comprising an amine group susceptible to protonation.Illustrative inorganic acids which form suitable salts includehydrochloric, hydrobromic, sulfuric and phosphoric acids, as well asmetal salts such as sodium monohydrogen orthophosphate and potassiumhydrogen sulfate. Illustrative organic acids that may form suitablesalts include mono-, di-, and tricarboxylic acids such as glycolic,lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric,citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylicacids, as well as sulfonic acids such as p-toluene sulfonic andmethanesulfonic acids. Such salts may exist in a hydrated, solvated orsubstantially anhydrous form. The selection of a suitable salt can bemade by a person skilled in the art. The formation of a desired acidaddition salt is, for example, achieved using standard techniques. Forexample, in an embodiment of the present disclosure, the neutralcompound is treated with the desired acid in a suitable solvent and thesalt which is thereby formed then isolated by filtration, extractionand/or any other suitable method.

A “base addition salt that is compatible with the treatment of subjects”is any non-toxic inorganic or organic salt of any acidic compound.Acidic compounds that form a base addition salt include, for example,compounds comprising a carboxylic acid group. Inorganic bases that mayform suitable salts include, without limitation, lithium, sodium,potassium, calcium, magnesium or barium hydroxide. Organic bases thatmay form suitable salts include, without limitation, aliphatic,alicyclic or aromatic organic amines such as methylamine, trimethylamineand picoline or ammonia. The selection of a suitable salt can be made bya person skilled in the art. The formation of a desired base additionsalt is, for example, achieved using standard techniques. For example,in an embodiment of the present disclosure, the neutral compound istreated with the desired base in a suitable solvent and the salt whichis thereby formed then isolated by filtration, extraction and/or anyother suitable method.

The terms “to treat”, “treating” and “treatment” and the like as usedherein and as is well understood in the art, means an approach forobtaining beneficial or desired results, including clinical results. Forexample, in the context of treating a viral infection, beneficial ordesired clinical results include, but are not limited to alleviation oramelioration of one or more symptoms of the viral infection,diminishment of the extent of the viral infection, stabilized (i.e., notworsening) of the viral infection, delay or slowing of the progressionof the viral infection, amelioration or palliation of the disease stateof the viral infection, diminishment of the reoccurrence of the viralinfection, and/or remission (whether partial or total) of the viralinfection, whether detectable or undetectable. “To treat”, “treating”and “treatment” and the like as used herein also include prophylactictreatment.

The compounds of the present disclosure are, for example, administeredto the subject or used in an “effective amount”.

As used herein, the term “effective amount” and the like means an amounteffective, at dosages and for periods of time necessary to achieve adesired result. For example, in the context of treating a viralinfection, an effective amount of a compound administered or used is anamount that, for example, reduces the viral infection compared to theviral infection without administration or use of the compound. Effectiveamounts may vary according to factors such as the disease state, age,sex, weight and/or species of the subject. The amount of a givencompound that will correspond to such an amount will vary depending uponvarious factors, such as the given compound, the pharmaceuticalformulation, the route of administration or use, the type of viralinfection being treated, the identity of the subject being treated, andthe like, but can nevertheless be routinely determined by one skilled inthe art.

II. Methods of Treatment and Uses

Cladoniamide A produced by actinomycete Streptomyces unicialis is activeagainst coronavirus and flavivirus. While S. unicialis also producesother cladoniamides, such as e.g. cladoniamide D, cladoniamide Dpossessed no activity against DENV and ZIKV. Similar results were alsofound for SARS-CoV-2. Cladoniamide E and cladoniamide F similarly showedno activity against ZIKV. Cladoniamide A was found to be a highlyeffective antiviral against the UK strain of SARS-CoV-2 (202012/01),with an ED₅₀ of about 800 pM. Cladoniamide A and cladoniamide C bothshowed anti-ZIKV activity in brain organoids infected with ZIKV.

Accordingly, the present disclosure includes a use of a compound of theFormula I or a pharmaceutically acceptable salt thereof for treatment ofa viral infection in a subject in need thereof:

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a use of a compound of the FormulaI or a pharmaceutically acceptable salt thereof for preparation of amedicament for treatment of a viral infection in a subject in needthereof:

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a compound of the Formula I or apharmaceutically acceptable salt thereof for use to treat a viralinfection in a subject in need thereof.

wherein each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a method of treating a viralinfection in a subject in need thereof, the method comprisingadministering a compound of the Formula I or a pharmaceuticallyacceptable salt thereof to the subject:

wherein each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

A person skilled in the art would readily appreciate in whichembodiments

represents a single bond and in which embodiments

represents a double bond. For example, the skilled person would readilyunderstand that when X¹ and/or X² is 0, the bond attaching the X¹ and/orthe X² to the remainder of the Formula I is a double bond whereas whenX¹ and/or X² is H or OR′ the bond attaching the X¹ and/or the X² to theremainder of the Formula I is a single bond. Similarly, the skilledperson would appreciate that when W¹ and W² are both absent, the bondconnecting the carbons to which W¹ and W² are attached is a double bondwhereas when W¹ and W² are each independently H, OR′, NR′ or SR′ thebond connecting the carbons to which W¹ and W² are attached is a singlebond.

In an embodiment, X¹ and X² are each independently O, H or OR′, whereinOR′ is H or C₁₋₆alkyl. In another embodiment, X¹ and X² are eachindependently OR′, wherein OR′ is H or C₁₋₆alkyl. In a furtherembodiment, X¹ and X² are both OH.

In an embodiment, Y¹ is N.

In an embodiment, Y² is NR′, S, O or CR′, wherein each R′ isindependently H or C₁₋₆alkyl. In another embodiment of the presentdisclosure, Y² is NR′, wherein R′ is H or C₁₋₆alkyl. In a furtherembodiment, Y² is NR′, wherein R′ is H.

In an embodiment, Y³ is N.

In an embodiment, W¹ and W² are absent. In another embodiment, W¹ and W²are each independently H, OR′, NR′ or SR′. In a further embodiment, W¹and W² are each OR′, wherein each R′ is independently H or C₁₋₆alkyl. Inanother embodiment, both W¹ and W² are OH.

In an embodiment, R¹ is H or unsubstituted C₁₋₆alkyl. In anotherembodiment, R¹ is C₁₋₄alkyl. In a further embodiment, R¹ is methyl.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring oran unsubstituted aromatic ring. In an embodiment, Z⁶ and Z⁷ togetherform an optionally substituted aromatic ring, and Z¹, Z², Z³, Z⁴, and Z⁵are each independently H, halo, optionally substituted alkyl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OR′, wherein R′ is C₁₋₆alkyl. In a further embodiment, Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is halo. Inanother embodiment, Z² is chloro. In a further embodiment, Z² is H.

In an embodiment, Z⁶ and Z⁷ together form a 6-membered aromatic ring. Inanother embodiment, Z⁶ and Z⁷ together form an unsubstituted, 6-memberedaromatic ring.

In an embodiment, R′ and R″ are each independently H or an optionallysubstituted C₁₋₁₀alkyl or C₁₋₁₀alkylene-aryl. In another embodiment, R′and R″ are each independently H or an optionally substituted C₁₋₆alkylor C₁₋₆alkylene-C₆₋₁₀aryl. In another embodiment, R′ is H. In anotherembodiment, R′ is alkyl. In another embodiment, R′ is C₁₋₁₀alkyl. In afurther embodiment, R′ is C₁₋₆alkyl. In another embodiment, R′ isC₁₋₄alkyl. In an embodiment, R′ is alkylene-aryl. In another embodiment,R′ is C₁₋₁₀alkylene-aryl. In a further embodiment, R′ isC₁₋₆alkyleneC₆₋₁₀aryl. In another embodiment, R″ is H. In anotherembodiment, R″ is alkyl. In another embodiment, R″ is C₁₋₁₀alkyl. In afurther embodiment, R″ is C₁₋₆alkyl. In another embodiment, R″ isC₁₋₄alkyl. In an embodiment, R″ is alkylene-aryl. In another embodiment,R″ is C₁₋₁₀alkylene-aryl. In a further embodiment, R″ isC₁₋₆alkyleneC₆₋₁₀aryl.

In an embodiment, the compound is a compound of Formula I(a):

wherein

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃; or

Z⁶ and Z⁷ together form an optionally substituted aromatic ring, and Z¹,Z², Z³, Z⁴, and Z⁵ are each independently H, halo, optionallysubstituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″,NR′COR″, OR′, SR′, CN, or CF₃;

R¹, R², R³, and R⁴ are each independently H or an optionally substitutedalkyl; and

R′ and R″ are each independently H, or an optionally substituted alkyl.

In an embodiment, R¹ is H or unsubstituted C₁₋₆alkyl. In anotherembodiment, R¹ is C₁₋₄alkyl. In a further embodiment, R¹ is methyl.

In an embodiment, R² and R⁴ are each independently H or C₁₋₆alkyl. Inanother embodiment, R² and R⁴ are both H.

In an embodiment, R³ is H or C₁₋₆alkyl. In another embodiment, R³ is H.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring oran unsubstituted aromatic ring. In an embodiment, Z⁶ and Z⁷ togetherform an optionally substituted aromatic ring, and Z¹, Z², Z³, Z⁴, and Z⁵are each independently H, halo, optionally substituted alkyl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OR′, wherein R′ is C₁₋₆alkyl. In a further embodiment, Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is halo. Inanother embodiment, Z² is chloro. In a further embodiment, Z² is H.

In an embodiment, Z⁶ and Z⁷ together form a 6-membered aromatic ring. Inanother embodiment, Z⁶ and Z⁷ together form an unsubstituted, 6-memberedaromatic ring.

In an embodiment, R′ and R″ are each independently H or an optionallysubstituted C₁₋₁₀alkyl or C₁₋₁₀alkylene-aryl. In another embodiment, R′and R″ are each independently H or an optionally substituted C₁₋₆alkylor C₁₋₆alkylene-C₆₋₁₀aryl. In another embodiment, R′ is H. In anotherembodiment, R′ is alkyl. In another embodiment, R′ is C₁₋₁₀alkyl. In afurther embodiment, R′ is C₁₋₆alkyl. In another embodiment, R′ isC₁₋₄alkyl. In an embodiment, R′ is alkylene-aryl. In another embodiment,R′ is C₁₋₁₀alkylene-aryl. In a further embodiment, R′ isC₁₋₆alkyleneC₆₋₁₀aryl. In another embodiment, R″ is H. In anotherembodiment, R″ is alkyl. In another embodiment, R″ is C₁₋₁₀alkyl. In afurther embodiment, R″ is C₁₋₆alkyl. In another embodiment, R″ isC₁₋₄alkyl. In an embodiment, R″ is alkylene-aryl. In another embodiment,R″ is C₁₋₁₀alkylene-aryl. In a further embodiment, R″ isC₁₋₆alkyleneC₆₋₁₀aryl.

In an embodiment, the compound is a compound of Formula III:

In an embodiment, the compound is cladoniamide A, ent-cladoniamide A ormixtures thereof. In another embodiment, the compound is cladoniamide A.In a further embodiment, the compound is ent-cladoniamide A. In anotherembodiment, the compound is a mixture of cladoniamide A andent-cladoniamide A.

In an embodiment, the compound is cladoniamide C.

In an embodiment, the compound or the pharmaceutically acceptable saltthereof is the compound. In another embodiment, the compound or thepharmaceutically acceptable salt thereof is the pharmaceuticallyacceptable salt of the compound.

In an embodiment, the viral infection is mosquito-borne. In anembodiment, the viral infection is a co-infection of two or moreviruses. In an embodiment, the viral infection is caused by one or morecoronavirus, flavivirus and/or influenza virus. In an embodiment, theviral infection is caused by one or more coronavirus and/or flavivirus.In another embodiment, the viral infection is caused by one or morecoronavirus. In an embodiment, the coronavirus is one or more of severeacute respiratory syndrome (SARS) coronavirus-1 (SARS-CoV-1), SARScoronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome (MERS)coronavirus (MERS-CoV) and human coronavirus 229E (HCoV-229E). Inanother embodiment, the coronavirus is severe acute respiratory syndrome(SARS) coronavirus-1 (SARS-CoV-1). In another embodiment, thecoronavirus is SARS coronavirus-2 (SARS-CoV-2). In a further embodiment,the SARS-CoV-2 is SARS-CoV-2 South Africa (501Y.V2), SARS-CoV-2 UK (VOC202012/01) or SARS-CoV-2 Nigeria. In an embodiment, the SARS-CoV-2 isSARS-CoV-2 South Africa (501Y.V2). In another embodiment, the SARS-CoV-2is SARS-CoV-2 UK (VOC 202012/01). In a further embodiment, theSARS-CoV-2 is SARS-CoV-2 Nigeria. In another embodiment, the coronavirusis Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV). Inanother embodiment, the coronavirus is human coronavirus 229E(HCoV-229E). In an embodiment, the viral infection is caused by one ormore flavivirus. In another embodiment, the flavivirus is one or more ofdengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV) and yellow fever virus(YFV). In another embodiment, the flavivirus is one or more of West Nilevirus (WNV), Powassan virus (POWV), Japanese encephalitis virus (JEV)and yellow fever virus (YFV). In an embodiment, the flavivirus is denguevirus (DENV). In an embodiment, the DENV is DENV-2, DENV-3, DENV-4 or acombination thereof. In another embodiment, the DENV is DENV-2. Inanother embodiment, the DENV is DENV-3. In another embodiment, the DENVis DENV-4. In another embodiment, the flavivirus is West Nile virus(WNV). In a further embodiment, the flavivirus is Zika virus (ZIKV). Inanother embodiment, the flavivirus is Powassan virus (POWV). In anotherembodiment, the flavivirus is Japanese encephalitis virus (JEV). In anembodiment, the flavivirus is yellow fever virus (YFV). In anotherembodiment, the viral infection is caused by one or more influenzavirus. In an embodiment, the influenza virus is one or more of H1N1influenza A virus, an H5 avian influenza A virus and an H7 avianinfluenza A virus. In another embodiment, the influenza virus is H1N1influenza A virus. In another embodiment, the influenza virus is an H5avian influenza A virus. In another embodiment, the influenza virus isan H7 avian influenza A virus.

In an embodiment, the subject is a human.

The present disclosure also includes a use of a compound of Formula I ora pharmaceutically acceptable salt thereof for inhibitingvacuolar-H⁺ATPase (V-ATPase):

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a compound of Formula I or apharmaceutically acceptable salt thereof for use to inhibitvacuolar-H⁺ATPase (V-ATPase):

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

The present disclosure also includes a method of inhibitingvacuolar-H⁺ATPase (V-ATPase) comprising contacting the V-ATPase with acompound of Formula I or a pharmaceutically acceptable salt thereof:

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl.

A person skilled in the art would readily appreciate in whichembodiments

represents a single bond and in which embodiments

represents a double bond. For example, the skilled person would readilyunderstand that when X¹ and/or X² is 0, the bond attaching the X¹ and/orthe X² to the remainder of the Formula I is a double bond whereas whenX¹ and/or X² is H or OR′ the bond attaching the X¹ and/or the X² to theremainder of the Formula I is a single bond. Similarly, the skilledperson would appreciate that when W¹ and W² are both absent, the bondconnecting the carbons to which W¹ and W² are attached is a double bondwhereas when W¹ and W² are each independently H, OR′, NR′ or SR′ thebond connecting the carbons to which W¹ and W² are attached is a singlebond.

In an embodiment, X¹ and X² are each independently O, H or OR′, whereinOR′ is H or C₁₋₆alkyl. In another embodiment, X¹ and X² are eachindependently OR′, wherein OR′ is H or C₁₋₆alkyl. In a furtherembodiment, X¹ and X² are both OH.

In an embodiment, Y¹ is N.

In an embodiment, Y² is NR′, S, O or CR′, wherein each R′ isindependently H or C₁₋₆alkyl. In another embodiment of the presentdisclosure, Y² is NR′, wherein R′ is H or C₁₋₆alkyl. In a furtherembodiment, Y² is NR′, wherein R′ is H.

In an embodiment, Y³ is N.

In an embodiment, W¹ and W² are absent. In another embodiment, W¹ and W²are each independently H, OR′, NR′ or SR′. In a further embodiment, W¹and W² are each OR′, wherein each R′ is independently H or C₁₋₆alkyl. Inanother embodiment, both W¹ and W² are OH.

In an embodiment, R¹ is H or unsubstituted C₁₋₆alkyl. In anotherembodiment, R¹ is C₁₋₄alkyl. In a further embodiment, R¹ is methyl.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring oran unsubstituted aromatic ring. In an embodiment, Z⁶ and Z⁷ togetherform an optionally substituted aromatic ring, and Z¹, Z², Z³, Z⁴, and Z⁵are each independently H, halo, optionally substituted alkyl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OR′, wherein R′ is C₁₋₆alkyl. In a further embodiment, Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is halo. Inanother embodiment, Z² is chloro. In a further embodiment, Z² is H.

In an embodiment, Z⁶ and Z⁷ together form a 6-membered aromatic ring. Inanother embodiment, Z⁶ and Z⁷ together form an unsubstituted, 6-memberedaromatic ring.

In an embodiment, R′ and R″ are each independently H or an optionallysubstituted C₁₋₁₀alkyl or C₁₋₁₀alkylene-aryl. In another embodiment, R′and R″ are each independently H or an optionally substituted C₁₋₆alkylor C₁₋₆alkylene-C₆₋₁₀aryl. In another embodiment, R′ is H. In anotherembodiment, R′ is alkyl. In another embodiment, R′ is C₁₋₁₀alkyl. In afurther embodiment, R′ is C₁₋₆alkyl. In another embodiment, R′ isC₁₋₄alkyl. In an embodiment, R′ is alkylene-aryl. In another embodiment,R′ is C₁₋₁₀alkylene-aryl. In a further embodiment, R′ isC₁₋₆alkyleneC₆₋₁₀aryl. In another embodiment, R″ is H. In anotherembodiment, R″ is alkyl. In another embodiment, R″ is C₁₋₁₀alkyl. In afurther embodiment, R″ is C₁₋₆alkyl. In another embodiment, R″ isC₁₋₄alkyl. In an embodiment, R″ is alkylene-aryl. In another embodiment,R″ is C₁₋₁₀alkylene-aryl. In a further embodiment, R″ isC₁₋₆alkyleneC₆₋₁₀aryl.

In an embodiment, the compound is a compound of Formula I(a):

wherein

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃; or

Z⁶ and Z⁷ together form an optionally substituted aromatic ring, and Z¹,Z², Z³, Z⁴, and Z⁵ are each independently H, halo, optionallysubstituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″,NR′COR″, OR′, SR′, CN, or CF₃;

R¹, R², R³, and R⁴ are each independently H or an optionally substitutedalkyl; and

R′ and R″ are each independently H, or an optionally substituted alkyl.

In an embodiment, R¹ is H or unsubstituted C₁₋₆alkyl. In anotherembodiment, R¹ is C₁₋₄alkyl. In a further embodiment, R¹ is methyl.

In an embodiment, R² and R⁴ are each independently H or C₁₋₆alkyl. Inanother embodiment, R² and R⁴ are both H.

In an embodiment, R³ is H or C₁₋₆alkyl. In another embodiment, R³ is H.

In an embodiment, Z⁶ and Z⁷ together form a substituted aromatic ring oran unsubstituted aromatic ring. In an embodiment, Z⁶ and Z⁷ togetherform an optionally substituted aromatic ring, and Z¹, Z², Z³, Z⁴, and Z⁵are each independently H, halo, optionally substituted alkyl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃.

In an embodiment, Z⁵ is OR′, wherein R′ is alkyl. In another embodiment,Z⁵ is OR′, wherein R′ is C₁₋₆alkyl. In a further embodiment, Z⁵ is OCH₃.

In an embodiment, Z¹, Z³ and Z⁴ are all H.

In an embodiment, Z² is halo or H. In another embodiment, Z² is halo. Inanother embodiment, Z² is chloro. In a further embodiment, Z² is H.

In an embodiment, Z⁶ and Z⁷ together form a 6-membered aromatic ring. Inanother embodiment, Z⁶ and Z⁷ together form an unsubstituted, 6-memberedaromatic ring.

In an embodiment, R′ and R″ are each independently H or an optionallysubstituted C₁₋₁₀alkyl or C₁₋₁₀alkylene-aryl. In another embodiment, R′and R″ are each independently H or an optionally substituted C₁₋₆alkylor C₁₋₆alkylene-C₆₋₁₀aryl. In another embodiment, R′ is H. In anotherembodiment, R′ is alkyl. In another embodiment, R′ is C₁₋₁₀alkyl. In afurther embodiment, R′ is C₁₋₆alkyl. In another embodiment, R′ isC₁₋₄alkyl. In an embodiment, R′ is alkylene-aryl. In another embodiment,R′ is C₁₋₁₀alkylene-aryl. In a further embodiment, R′ isC₁₋₆alkyleneC₆₋₁₀aryl. In another embodiment, R″ is H. In anotherembodiment, R″ is alkyl. In another embodiment, R″ is C₁₋₁₀alkyl. In afurther embodiment, R″ is C₁₋₆alkyl. In another embodiment, R″ isC₁₋₄alkyl. In an embodiment, R″ is alkylene-aryl. In another embodiment,R″ is C₁₋₁₀alkylene-aryl. In a further embodiment, R″ isC₁₋₆alkyleneC₆₋₁₀aryl.

In an embodiment, the compound is a compound of Formula III:

In an embodiment, the compound is cladoniamide A, ent-cladoniamide A ormixtures thereof. In another embodiment, the compound is cladoniamide A.In a further embodiment, the compound is ent-cladoniamide A. In anotherembodiment, the compound is a mixture of cladoniamide A andent-cladoniamide A.

In an embodiment, the compound is cladoniamide C.

In an embodiment, the compound or the pharmaceutically acceptable saltthereof is the compound. In another embodiment, the compound or thepharmaceutically acceptable salt thereof is the pharmaceuticallyacceptable salt of the compound.

In an embodiment, the inhibition of V-ATPase is in a subject.

In an embodiment, the subject is a human.

In an embodiment, the subject suffers from a viral infection.

In an embodiment, the viral infection is mosquito-borne. In anembodiment, the viral infection is a co-infection of two or moreviruses. In an embodiment, the viral infection is caused by one or morecoronavirus, flavivirus and/or influenza virus. In an embodiment, theviral infection is caused by one or more coronavirus and/or flavivirus.In another embodiment, the viral infection is caused by one or morecoronavirus. In an embodiment, the coronavirus is one or more of severeacute respiratory syndrome (SARS) coronavirus-1 (SARS-CoV-1), SARScoronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome (MERS)coronavirus (MERS-CoV) and human coronavirus 229E (HCoV-229E). Inanother embodiment, the coronavirus is severe acute respiratory syndrome(SARS) coronavirus-1 (SARS-CoV-1). In another embodiment, thecoronavirus is SARS coronavirus-2 (SARS-CoV-2). In a further embodiment,the SARS-CoV-2 is SARS-CoV-2 South Africa (501Y.V2), SARS-CoV-2 UK (VOC202012/01) or SARS-CoV-2 Nigeria. In an embodiment, the SARS-CoV-2 isSARS-CoV-2 South Africa (501Y.V2). In another embodiment, the SARS-CoV-2is SARS-CoV-2 UK (VOC 202012/01). In a further embodiment, theSARS-CoV-2 is SARS-CoV-2 Nigeria. In another embodiment, the coronavirusis Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV). Inanother embodiment, the coronavirus is human coronavirus 229E(HCoV-229E). In an embodiment, the viral infection is caused by one ormore flavivirus. In another embodiment, the flavivirus is one or more ofdengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Powassanvirus (POWV), Japanese encephalitis virus (JEV) and yellow fever virus(YFV). In another embodiment, the flavivirus is one or more of West Nilevirus (WNV), Powassan virus (POWV), Japanese encephalitis virus (JEV)and yellow fever virus (YFV). In an embodiment, the flavivirus is denguevirus (DENV). In an embodiment, the DENV is DENV-2, DENV-3, DENV-4 or acombination thereof. In another embodiment, the DENV is DENV-2. Inanother embodiment, the DENV is DENV-3. In another embodiment, the DENVis DENV-4. In another embodiment, the flavivirus is West Nile virus(WNV). In a further embodiment, the flavivirus is Zika virus (ZIKV). Inanother embodiment, the flavivirus is Powassan virus (POWV). In anotherembodiment, the flavivirus is Japanese encephalitis virus (JEV). In anembodiment, the flavivirus is yellow fever virus (YFV). In anotherembodiment, the viral infection is caused by one or more influenzavirus. In an embodiment, the influenza virus is one or more of H1N1influenza A virus, an H5 avian influenza A virus and an H7 avianinfluenza A virus. In another embodiment, the influenza virus is H1N1influenza A virus. In another embodiment, the influenza virus is an H5avian influenza A virus. In another embodiment, the influenza virus isan H7 avian influenza A virus.

In an embodiment, the compound is administered or used in apharmaceutical composition comprising the compound and optionally apharmaceutically acceptable carrier.

The compound can be administered to a subject or used in a variety offorms depending on the selected route of administration or use, as willbe understood by those skilled in the art. In an embodiment, thecompound is administered to the subject, or used, by oral (includingbuccal) or parenteral (including intravenous, intraperitoneal,subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary,intrathecal, rectal, topical, patch, pump and transdermal)administration or use and the compound formulated accordingly. Forexample, the compound is administered or used in an injection, in aspray, in a tablet/caplet, in a powder, topically, in a gel, in drops,by a patch, by an implant, by a slow release pump or by any othersuitable method of administration or use, the selection of which can bemade by a person skilled in the art.

In an embodiment, the compound is orally administered or used, forexample, with an inert diluent or with an assimilable edible carrier, orenclosed in hard- or soft-shell gelatin capsules, or compressed intotablets, or incorporated directly with the food of the diet. In anembodiment, for oral therapeutic administration or use, the compound isincorporated with excipient and administered or used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Oral dosage forms alsoinclude modified release, for example immediate release andtimed-release, formulations. Examples of modified-release formulationsinclude, for example, sustained-release (SR), extended-release (ER, XR,or XL), time-release or timed-release, controlled-release (CR), orcontinuous-release (CR or Contin), employed, for example, in the form ofa coated tablet, an osmotic delivery device, a coated capsule, amicroencapsulated microsphere, an agglomerated particle, e.g., asmolecular sieving type particles, or, a fine hollow permeable fiberbundle, or chopped hollow permeable fibers, agglomerated or held in afibrous packet. Timed-release compositions can be formulated, e.g.liposomes or those wherein the compound is protected with differentiallydegradable coatings, such as by microencapsulation, multiple coatings,etc.

In another embodiment, the compound is administered or usedparenterally. Solutions of the compound are, for example, prepared inwater optionally mixed with a surfactant such as hydroxypropylcellulose.In a further example, dispersions are prepared in glycerol, liquidpolyethylene glycols, dimethyl sulfoxide (DMSO) or mixtures thereof withor without alcohol, or in oils. Pharmaceutical forms suitable forinjectable administration or use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. A person skilled in the artwould know how to select and to prepare suitable formulations.

Treatment methods or uses comprise administering to a subject or use ofan effective amount of the compound, optionally consisting of a singleadministration or use, or alternatively comprising a series ofadministrations or uses. For example, the compound is administered orused at least once a week. However, in another embodiment, the compoundis administered to the subject or used from one time per three weeks, orone time per week to once daily for a given treatment or use. In anotherembodiment, the compound is administered or used 2, 3, 4, 5 or 6 timesdaily. The length of the treatment period or use depends on a variety offactors, such as the severity and/or type of the viral infection, theage of the subject, the concentration of the compound in a formulation,the activity of the compound and/or a combination thereof. It will alsobe appreciated that the effective amount of a compound used for thetreatment or use may increase or decrease over the course of aparticular treatment regime or use. Changes in dosage may result andbecome apparent by standard diagnostic assays known in the art. In someinstances, chronic administration or use is required. For example, thecompound is administered or used in an amount and for a durationsufficient to treat the subject.

The compound may be administered or used alone or in combination withother therapeutic agents useful for treating a viral infection. Whenadministered or used in combination with other known therapeutic agents,it is an embodiment that the compound is administered or usedcontemporaneously with those therapeutic agents. As used herein, theterm “contemporaneous” in reference to administration of two substancesto a subject or use means providing each of the two substances so thatthey are both biologically active in the individual at the same time.The exact details of the administration or use will depend on thepharmacokinetics of the two substances in the presence of each other,and can include administering or using the two substances within a fewhours of each other, or even administering or using one substance within24 hours of administration or use of the other, if the pharmacokineticsare suitable. Design of suitable dosing regimens is routine for oneskilled in the art. In particular embodiments, two substances will beadministered or used substantially simultaneously, i.e., within minutesof each other, or in a single composition that contains both substances.It is a further embodiment that a combination of the two substances isadministered to a subject or used in a non-contemporaneous fashion.

The dosage of the compound can vary depending on many factors such asthe pharmacodynamic properties of the compound, the mode ofadministration or use, the age, health and weight of the subject, thetype of viral infection, the nature of and/or extent of the symptoms ofthe viral infection, the frequency of the treatment or use and the typeof concurrent treatment or use, if any, and the clearance rate of thecompound in the subject. One of skill in the art can determine theappropriate dosage based on the above factors. In an embodiment, thecompound is administered or used initially in a suitable dosage that isoptionally adjusted as required, depending on the clinical response. Asa representative example, oral dosages of the compound will range fromless than 1 mg per day to 1000 mg per day for a human adult or ananimal. In an embodiment of the present disclosure, the pharmaceuticalcompositions are formulated for oral administration or use and thecompounds are, for example in the form of tablets containing 0.001,0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0, 40.0,50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg ofactive ingredient per tablet. In an embodiment, the compound isadministered or used in a single daily dose or the total daily dose maybe divided into e.g. two, three or four daily doses.

The present disclosure also includes a use of a compound of Formula I ora pharmaceutically acceptable salt thereof as defined herein to assessactivity of a virus (e.g. a virus causing a viral infection as definedherein) against a vacuolar-H⁺ATPase (V-ATPase).

III. Compounds, Compositions and Methods of Preparation

The present disclosure also includes a compound of Formula I(b):

wherein

each

independently represents a single or double bond;

Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, halo, alkyl,cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′,SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl,alkylene-aryl, aryl and heteroaryl are optionally substituted; or

one or more of Z¹ and Z², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷together form an optionally substituted carbocyclic or heterocyclicring, and the remainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are eachindependently H, halo, alkyl, cycloalkyl, alkylene-aryl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″,NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN orhaloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl, aryl andheteroaryl are optionally substituted;

X¹ and X² are each independently O, H or OR′;

Y¹ is N or CR′;

Y² is NR′, S, O or CR′;

Y³ is N or CR′;

W¹ and W² are both absent or are each independently H, OR′, NR′ or SR′;

R¹ is H or an optionally substituted alkyl; and

R′ and R″ are each independently H or an optionally substituted alkyl oralkylene-aryl,

or a pharmaceutically acceptable salt thereof, with the proviso thatsaid compound is not exactly:

or wherein R⁵ is C₁₋₄alkyl, X² is selected from H and halo, and R¹ isselected from H, halo and OH.

In an embodiment, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, X¹, X², Y¹, Y², Y³, W¹,W², R¹, R′ and R″ are as defined herein for the compound of Formula I solong as the compound is not exactly any of the structures shownhereinabove in the proviso for the compound of Formula I(b).

The present disclosure also includes a compound of Formula II:

wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, and X⁸ are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′S₂,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃;

R¹, R², R³, R⁴, and R⁵ are each independently H or an optionallysubstituted alkyl; and

R′ and R″ are each independently H, or an optionally substituted alkyl;

or a pharmaceutically acceptable salt thereof, with the proviso thatsaid compound is not exactly:

or wherein R⁵ is C₁₋₄alkyl, X² is selected from H and halo, and R¹ isselected from H, halo and OH.

The present disclosure also includes a composition comprising one ormore compounds (e.g. a compound of Formula I(b), Formula II or FormulaII(a) as defined herein) and a carrier. The compounds are optionallyformulated into pharmaceutical compositions for administration tosubjects or use in a biologically compatible form suitable foradministration and use in vivo. Accordingly, the present disclosurefurther includes a pharmaceutical composition comprising one or morecompounds (e.g. a compound of Formula I(b), Formula II or Formula II(a)as defined herein) and a pharmaceutically acceptable carrier.

In an embodiment, the compound is a compound of Formula II and thecompound is prepared, for example, by a method comprising the reactionsshown in Scheme 1. In the compounds of Formulae II and IV to VIII, X¹-X⁸and R¹-R⁵ are as defined herein. A skilled person could readily useand/or adapt such a method to prepare a compound of Formula I, FormulaI(a), Formula I(b), Formula II(a), Formula III, Formula III(a) orFormula III(b).

Various alternative embodiments and examples are described herein. Theseembodiments and examples are illustrative and should not be construed aslimiting the scope of the invention. The following are non-limitingexamples of the present disclosure:

EXAMPLES Materials and Methods

The procedures described herein are given for the purposes of exampleand illustration only and should not be considered to limit the spiritor scope of the invention.

The cladoniamides used in these studies were obtained by fermentationand isolation based on a previous literature report by Williams et al.(2008). Cladoniamides may, for example, also be chemically synthesizedor biosynthesized. For example, the chemical synthesis of cladoniamide Amay follow a literature report by Kimura et al. (2012). The biosynthesisof cladoniamides may be based on a literature report by Du et al.(2014).

Cell culture. Vero E6 cells (ATCC #CCL-81), A549 cells (ATCC #CCL-185,or acquired from Synthego), and U87 MG cells (ATCC #HTB-14, or acquiredfrom Synthego) were maintained in Minimum Essential Medium (MEM; Gibco)supplemented with 10% fetal bovine serum (FBS; Gibco), 1% non-essentialamino acids (Gibco), and 1 mM sodium pyruvate (Gibco). Huh-7.5.1 cellsand Huh-7.5 replicon-containing cells were cultured in Dulbecco'sModified Eagle's Medium (DMEM; Gibco) supplemented with 10% FBS (Gibco),10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES;Gibco), and 1% non-essential amino acids (Gibco). Thereplicon-containing cells were maintained in the presence of 0.3 mg/mLgentamicin (Gibco). All incubations were performed at 37° C. in thepresence of 5% CO₂ unless otherwise noted.

Viruses. ZIKV: ZIKV strain PRVABC59 (Puerto Rico/2015) (ATCC #VR-1843)was used in these studies, specifically three separate stocks generatedin Vero E6 cells with titres of 1.35×10⁸ pfu/mL, 1.0×10⁷ pfu/mL, and2.9×10⁶ pfu/mL respectively. DENV strains: DENV-2 strain NGC, DENV-3strain H-87, and DENV-4 strain H-241 were kindly provided by NationalMicrobiology Laboratory, Winnipeg, MB, Canada.

Semisolid overlay plaque assays. Vero E6 cell monolayers were seeded in12 well plates (Sarstedt) and incubated for 24 h. Supernatants of ZIKVinfected cells were serially diluted starting at 1:100 and used toinoculate the cells for 1 h. Virus was then removed and plaque assaycultures were overlaid with 1% low melt agarose (Gibco) in minimumessential media (MEM; Gibco) supplemented with 2% heat inactivated FBS(Sigma-Aldrich Corp.), 1% non-essential amino acids (Gibco) and 1 mMsodium pyruvate (Gibco). The agarose overlay was melted and maintainedat 42° C. before adding to the cells. Plaques were fixed after 5 dayswith 3.7% formaldehyde in PBS (Gibco) for 1 h, after which the overlaywas removed, and plaques were visualized by staining with 1% crystalviolet (Sigma-Aldrich Corp.) in 20% methanol.

Liquid overlay plaque assays. Vero E6 cell monolayers were seeded in 12well plates (Sarstedt) and incubated for 24 h. Supernatants of ZIKVinfected cells were serially diluted starting at 1:100 and used toinoculate the cells for 1 h. Virus was then removed and plaque assaycultures were overlaid with 1% microcrystalline cellulose (Avicel™CL-611; DuPont) in MEM (Gibco) supplemented with 2% FBS (Gibco), 1%non-essential amino acids (Gibco) and 1 mM sodium pyruvate (Gibco). Theoverlay was removed, and plaques were fixed after 3 days with 3.7%formaldehyde in PBS (Gibco) for 1 h. Plaques were visualized by stainingwith 1% crystal violet (Sigma-Aldrich Corp.) in 20% methanol.

Example 1: Anti-ZIKV Activity

An MTS assay was performed to determine the cytotoxicity concentration50% (CC₅₀) of cladoniamide A (CA) in human A549 cells, a cell line fromhuman lung carcinoma. Various concentrations of CA were added tocultured A549 cells and incubated (37° C., 5% CO₂) for 48 h. Cellviability was then measured by an MTS assay kit (CellTiter 96™ AqueousOne Solution cell proliferation assay, Promega) according to themanufacturer's instructions. Curve fitting was performed in Igor Pro(WaveMetrics) using a hyperbolic fit algorithm (FIG. 1 ). The CC₅₀ of CAin A549 cells was determined to be 54±5 μM. An MTS assay was alsoperformed to determine the CC₅₀ of CA in human U87 MG cells, a cell linefrom human brain. Various concentrations of CA were added to culturedU87 MG cells and incubated (37° C., 5% CO₂) for 48 h. Cell viability wasthen measured by an MTS assay kit (CellTiter 96 Aqueous One Solutioncell proliferation assay, Promega) according to the manufacturer'sinstructions. Curve fitting was performed in Igor Pro (WaveMetrics)using a hyperbolic fit algorithm (FIG. 2 ). The CC₅₀ of CA in U87 MGcells was determined to be 42±12 μM.

Cultured A549 and U87 MG cells were treated with CA (250 nM),cladoniamide D (CD; 15 μM), or PF-429242 (Hyrina 2017; 15 μM) for 1 hbefore being washed with PBS and inoculated with ZIKV at multiplicity ofinfection (MOI) 1.0 for 1 h. Following the removal of the inoculum,cells were incubated in fresh media for 3 d before the supernatant wascollected, clarified, and ZIKV infectivity was measured by semisolidoverlay plaque assay. Hatched bars representing limit of quantitation(LOQ) of the plaque assay are shown where values below LOQ were obtained(FIG. 3 ). There was a greater than 4-log reduction in ZIKV titer whenA549 and U87 MG cells were treated with CA.

Cultured A549 cells were treated with various concentrations of CA orPF-429242 for 24 h before being washed with PBS and inoculated with ZIKV(ZIKV strain VR 1843, Puerto Rico, 2015; ATCC) at MOI 1.0 for 1 h.Following the removal of the inoculum, cells were incubated in freshmedia for 2 d before the supernatant was collected, clarified, andsecreted NS1 protein abundance was measured by an ELISA kit (BioFrontTechnologies) according to the manufacturer's instructions. Curvefitting was performed in Igor Pro (WaveMetrics) using a hyperbolic fitalgorithm (FIG. 4 ; upper and middle plots) and IC₅₀ and TI valuesobtained. IC₅₀ and TI values were also obtained for CA via plaque assay(PFU; FIG. 4 , lower plot). FIG. 4 (upper plot) is an ED₅₀ curve for thePF inhibitor based on the detection of secreted Non-Structural protein 1(sNS1), secreted viral biomarker. sNS1 was detected using an Elisa test(commercially available). FIG. 4 (middle plot) is an ED₅₀ curve for CAbased on the detection of secreted Non-Structural protein 1 (sNS1),secreted viral biomarker. sNS1 was detected using an Elisa test(commercially available).

Bottom: ED50 curve for CA based on the detection of secreted infectiousvirus particles, as viral biomarker. Titer of the infectious virusparticles (Plaque forming units) determined using plaque assays. Theresults of these studies are summarized in Table 1.

TABLE 1 CA vs PF as anti-ZIKV agents in human A549 cells. IAA CC₅₀ (μM)IC₅₀ (μM) TI PF 69 13* 5.5 CA 54 0.08* 675 CA 54 0.110** 490 *ELISA(sNS1); **Plaque assay (PFU).

Cultured Huh-7.5.1 cells and Huh-7.5 ZIKV replicon-containing cells (Xie2016) were treated with various concentrations of CA or putative ZIKVreplication inhibitor NITD-008 (Deng 2016) for 48 h. Cells were thenwashed with PBS and ViviRen™ Live Cell Substrate (Promega) was added toa final concentration of 30 μM. Following incubation for 2 min at roomtemperature, luminescence was measured. Curve fitting was performed inIgor Pro (WaveMetrics) using a sigmoidal fit algorithm (FIG. 5 ). As canbe seen from the results in FIG. 5 , in contrast to NITD-008, CA is nota ZIKV replication inhibitor.

Cultured A549 cells were inoculated with ZIKV for 1 h at MOI 1.0, andmaintained for 2 d under different experimental conditions. For “Fulllifecycle” 250 nM CA was added following the inoculation and maintainedin the medium for 2 d; for “Pre-replication” 250 nM CA was addedfollowing the inoculation and maintained in the medium for 6 h, afterwhich the cells were washed with PBS and the medium was changed; and for“Post-replication” 250 nM CA was added to the culture medium 12 hourspost infection (hpi) and maintained until 24 hpi, after which the cellswere washed with PBS and the medium was changed. Limit of detection(LOD) of the plaque assay is indicated in FIG. 6 .

Cladoniamide A demonstrated good activity against ZIKV in vitro (FIG. 1-FIG. 6 ). While cladoniamide A dramatically inhibited the production ofinfectious ZIKV virions at 50 nm, cladoniamide D did not show anyinhibitory effect (FIG. 3 ).

A549 cells were seeded onto Coming tissue culture treated 96 well platesat 6500 cells/well (100 μL) and incubated at 37 degrees Celsius and 5%CO₂ for 24 h. CA, CD, cladoniamide E (CE) and cladoniamide F (CF) werediluted in MEM complete media to concentrations of 15 μM. The dilutedCA, CD, CE or CF was added to the respective wells and cells wereincubated at 37 degrees Celsius and 5% CO₂ for 1 h. After 1 h, cellswere washed once with PBS to remove the CA, CD, CE or CF and were theninfected with ZIKV at an MOI of 1. Cells were incubated with ZIKV for 48h at 37 degrees Celsius and 5% CO₂. Cells were fixed with 3.7%formaldehyde in PBS then labeled with the J2 anti-dsRNA IgG2a monoclonalantibody (Scicons) at a concentration of 1:500. Cells were then labeledwith the goat anti-mouse Alexa Fluor™ 488 (ThermoFisher) at 1:2000.Labeled 96 well plates were imaged and quantified using the CellomicsArrayScan VTI. CA demonstrated good activity against ZIKV whereas CD, CEand CF did not show any inhibitory effect (FIG. 7 ).

Example 2: Anti-DENV Activity

A549 cells were seeded onto Coming tissue culture treated 96 well platesand incubated at 37 degrees Celsius and 5% CO₂ for 24 h. CA was dilutedin MEM complete media to concentrations of 1 μM, 5 μM, 10 μM, 20 μM,including a 0 μM condition. Diluted CA was added to each well and cellswere incubated at 37 degrees Celsius and 5% CO₂ for 1 h. After 1 h,cells were washed once with PBS to remove CA and were then infected withDENV-2, -3 or -4 at an MOI of 1. Cells were incubated with DENV for 48 hat 37 degrees Celsius and 5% CO₂. Cells were fixed with 3.7%formaldehyde in PBS then labeled with the J2 anti-dsRNA IgG2a monoclonalantibody (Scicons) at a concentration of 1:500. Cells were then labeledwith the goat anti-mouse Alexa Fluor™ 488 (ThermoFisher) at 1:2000 andHoechst. Labeled 96 well plates were imaged and quantified using theCellomics ArrayScan VTI. Curve fitting was performed in Igor Pro(WaveMetrics) using a hyperbolic fit algorithm. Cladoniamide Ademonstrated good activity against Dengue virus 2 (FIG. 8 , upperplots), Dengue virus 3 (FIG. 8 , middle plots) and Dengue virus 4 (FIG.8 , lower plots) in vitro. Table 2 provides a summary of the results. Adrug is generally considered to have a good safety profile if its TI isgreater than 10 (Muller & Milton 2012).

TABLE 2 CA is a pan-serotype inhibitor against DENV in human A549 cells.DENV IC₅₀ (μM) TI DENV-2 2.3* 24 DENV-3 1.5* 36 DENV-4 3.2* 17 *IC₅₀values calculated based on intracellular dsRNA (Cellomics); CC₅₀: 54 ± 5μM.

FIG. 9 shows similar results for Huh 7.5.1 cells (left plots) and U87cells (right plots), against Dengue virus 2 (upper plots), Dengue virus3 (middle plots) and Dengue virus 4 (lower plots) indicting thebroad-spectrum anti-flaviviral activity of CA.

Example 3: V-ATPase Inhibitor Activity

The crystal structure of the E. hirae ntpK V-type Na⁺-ATPase is known(PDB 2BL2) (Murata 2005). E. hirae ntpK is homologous to H. sapiensATP6V0C (Chang 2014). In the top image of FIG. 9 , residues for whichmutations associated with CA resistance have been found (▪); andbafilomycin A1 resistance-associated mutations (●) are indicated (Chang2014). FIG. 10 (lower left) is a schematic showing Vma3 topology and keyamino acids, with the CA potential binding site indicated (dottedlines). The protein structure 10 of hVma3 (ATP6V0C) was visualized inPyMol (version 1.3; Schrodinger LLC) with one chain of the complexhighlighted (FIG. 10 , lower right). Referring to FIG. 10 (lower right)a CA potential binding site (5 specific amino acids) 12 and BAf1 bindingsite 14 are also indicated.

Cultured A549 cells were treated with 1 μM CD, or 250 nM CA or theV-ATPase specific inhibitor bafilomycin A1 (100 nm) for 6 h. Afterremoving the culture medium and washing the cells with Hanks' balancedsalt solution (HBSS), a 1:4000 dilution of acridine orange (2% solution,Polysciences Inc.) in HBSS was added to cells and incubated for 10 min.Cells were then washed again with HBSS and analyzed by fluorescencemicroscopy. In color images, a red color corresponds to the protonatedversion of the molecule, which permeates acidic organelles and becomessequestered following protonation. One representative of 3 biologicalreplicates is shown. FIG. 11 and FIG. 12 show results of the 6-hourtreatment of A549 cells with the V-ATPase specific inhibitor bafilomycinA1 and cladoniamide A as well as cladoniamide D. Cladoniamide A, but notcladoniamide D, lead to intracellular pH dysregulation and inhibitedvital staining with acridine orange of the intracellular organelles ofhuman A549 cells. FIG. 13 shows results of the time course of treatmentof A549 cells with bafilomycin A1, CA and CD.

Example 4: Anti-HcoV-229E Activity

CA pre-treatment: Cladoniamide A was added to non-infected Huh-7 cells(3 h). Cells were washed with PBS and inoculated with human coronavirus229E (HCoV-229E) for 3 hours, then HCoV-229E was removed and the cellswere washed with PBS once more. 48 hours post-infection, the cells werefixed and the intra-cellular viral dsRNA was quantified (HCS CX5system). The results are shown in FIG. 14 (top). CA addedpost-infection: Huh-7 cells were inoculated with HCoV-229E for 3 hours.Then HCoV-229E was removed and cells were washed with PBS. CladoniamideA was added to these infected cells. 48 hours post-infection, the cellswere fixed and the intra-cellular viral dsRNA was quantified (HCS CX5system). The results are shown in FIG. 14 (bottom). The experiment wasconducted in triplicate, and the average EC₅₀ value from these threeindependent experiments was EC₅₀=15 nM. These results show CA is ananomolar inhibitor of HCoV-229E.

Example 5: Anti-SARS-CoV activity

All infections were carried out in a Biosafety Level 3 (BSL3) facility(UBC FINDER) in accordance with the Public Health Agency of Canada andUBC FINDER regulations. SARS-CoV-2 (SARS-COV-2/Canada/VIDO-01/2020) waspassaged in Vero E6 cells. For experiments, passage three of the viruswas used with a determined viral titer of 1.5×10⁷ plaque forming units(PFU)/mL. Calu-3 cells were seeded at a concentration of 10,000cells/well in 96-well plates the day before infection. SARS-CoV-2 stockswere diluted in cell-specific media to a multiplicity of infection (MOI)of 2. Cells were pretreated with compounds for three hours and thenincubated with the virus for 2 days, followed by fixation of the cellswith 3.7% formalin for 30 min to inactivate the virus. The fixative wasremoved, cells were washed with PBS, and permeabilized with 0.1% TritonX-100 for 5 minutes, followed by immunostaining with the mouse primaryantibody J2 (dsRNA) and rabbit primary antibody HL344 (SARS-CoV-2nucleocapsid) at working dilutions of 1:1000 for 1 hour at roomtemperature. Secondary antibodies were used at a 1:2000 dilution andincluded the goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbitIgG Alexa Fluor 555 with the nuclear stain Hoechst 33342 at 1 μg/mL for1 hour at room temperature in the dark. After washing with PBS, plateswere kept covered in aluminum foil at 4° C. until imaging on a HighContent Screening (HCS) platform (CellInsight™ CX7 HCS, Thermo FisherScientific) with a 10X objective, or a EVOS™ M7000 Imaging System(Thermo Fisher Scientific) with a 20X or 40X objective.

FIG. 15 shows data for CA activity against SARS-CoV-2. ED₅₀ values 2 ofnM and 5 nM were obtained based on intracellular viral RNA (FIG. 15 ,upper plot) or intracellular SARS-CoV-2 nucleocapsid protein (FIG. 15 ,lower plot), respectively. In contrast, no antiviral effect of CD inHuman Caco-2 cells against SARS-CoV-2 was detected (FIG. 15 ). In each,3 hours pre-treatment of the cells was carried out with CA or CD beforeadding the virus. These results were in line with the data obtained forZIKV and DENV. While not wishing to be limited by theory, the mechanismis V-ATPase regulation of intracellular pH.

FIG. 16 shows results for SARS-CoV-2 Variants; namely SARS-CoV-2 Wuhanstrain from VIDO (SB); SARS-CoV-2 England (VOC 202012/01) from BC-CDC(UK); SARS-CoV-2 South Africa (501Y.V2) from BC-CDC (SA); and SARS-CoV-2Nigeria (484) (Nigeria). CA was observed to be a very potent antiviralfor the SARS-CoV-2 Variants of Concern (VOCs) tested, namely SARS-CoV-2South Africa (501Y.V2), SARS-CoV-2 UK (VOC 202012/01) and SARS-CoV-2Nigeria. The IC₅₀ values are summarized in Table 3. Importantly, CA wasfound to be an 800 pM inhibitor of the UK variant (ED₅₀ 800 pM). Reportshave suggested that the UK coronavirus variant is 70 percent moretransmissible than the other variants. The UK variant has also beenreported to be 64 percent deadlier than previous strains. These resultsalso underline, for example, that CA and similar compounds may representanew molecular tool to study CoV biology. For example, CA and similarcompounds could permit the investigation of potential discrepancies inthe viral hijacking of host-cell V-ATPase for new VOCs and/or unraveldifferences between the VOCs for usurping V-ATPase during the CoVinfection. Further, such compounds may have broader applications incombating other widespread human enveloped viruses hijacking V-ATPasesto support their lifecycles including pandemic H1N1 influenza A virusand highly pathogenic H5/H7 avian influenza A viruses.

TABLE 3 summary of IC₅₀ values for SARS-CoV-2 variants. SB2 UK SANigeria IC₅₀ (nM) 57.69 0.8010 12.25 12.17

Example 6: Anti-ZIKV Activity in Brain Organoids

FIG. 17 is a schematic showing the general method 20 used in Example 6.Referring to FIG. 17 , following growth of the brain organoids inSTEMdiff™ media 22 (4 organoids/well), the media was aspirated 24. Then,500 μL ZIKV at the desired MOI in fresh media was added 26 and the brainorganoids incubated at 37° C. for 3 hours with gentle shaking 28. Thebrain organoids were then washed with 2×1 mL PBS 30, and fresh media andCA or cladoniamide C (CC) added to the desired concentration 32 prior toincubation for the desired time at 37° C. 34. The supernatant wascollected and frozen at −86° C. 36 prior to analysis and the brainorganoids transferred to an Eppendorf with paraformaldehyde for electronmicroscopy 38.

FIG. 18 is a plot showing the anti-ZIKV activity of cladoniamide A(incubation with 1 μM CA for 72 hours; 2 biological replicates, eachwith 4 organoids/well) using four brain organoid systems. There was agreater than 4-log reduction in extracellular ZIKV titer when treatedwith CA. A toxicity assay (CellTiter Glo™ 3D cell viability assay kit)showed CA is not toxic at 1 μM to brain organoids (FIG. 19 ). FIGS. 20and 21 show plots showing the anti-ZIKV activity of CA and CC(incubation with 10 μM CA or CC for 69 hours, MOI 1; 4 technicalreplicates as there were 4 organoids/cell).

While the disclosure has been described with reference to what arepresently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the present disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims. Although various embodiments of theinvention are disclosed herein, many adaptations and modifications maybe made within the scope of the invention in accordance with the commongeneral knowledge of those skilled in this art. Such modificationsinclude the substitution of known equivalents for any aspect of theinvention in order to achieve the same result in substantially the sameway.

REFERENCES

Citation of references herein is not an admission that such referencesare prior art to an embodiment of the present invention. The referenceto any prior art in this specification is not, and should not be takenas, an acknowledgement or any form of suggestion that that prior artforms part of the common general knowledge in Canada or any othercountry. The invention includes all embodiments and variationssubstantially as hereinbefore described and with reference to theexamples and drawings. Titles, headings, or the like are provided toenhance the reader's comprehension of this document, and should not beread as limiting the scope of the present invention. All publications,patents and patent applications are herein incorporated by reference intheir entirety to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety. Where a term in thepresent application is found to be defined differently in a documentincorporated herein by reference, the definition provided herein is toserve as the definition for the term.

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1. A use of a compound of the Formula I or a pharmaceutically acceptablesalt thereof for treatment of a viral infection in a subject in needthereof:

wherein each

independently represents a single or double bond; Z¹, Z², Z³, Z⁴, Z⁵,Z⁶, and Z⁷ are each independently H, halo, alkyl, cycloalkyl,alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′,CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′,SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl,aryl and heteroaryl are optionally substituted; or one or more of Z¹ andZ², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷ together form anoptionally substituted carbocyclic or heterocyclic ring, and theremainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H,halo, alkyl, cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″,NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″,OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl,cycloalkyl, alkylene-aryl, aryl and heteroaryl are optionallysubstituted; X¹ and X² are each independently O, H or OR′; Y¹ is N orCR′; Y² is NR′, S, O or CR′; Y³ is N or CR′; W¹ and W² are both absentor are each independently H, OR′, NR′ or SR′; R¹ is H or an optionallysubstituted alkyl; and R′ and R″ are each independently H or anoptionally substituted alkyl or alkylene-aryl.
 2. A use of a compound ofthe Formula I or a pharmaceutically acceptable salt thereof forpreparation of a medicament for treatment of a viral infection in asubject in need thereof:

wherein each

independently represents a single or double bond; Z¹, Z², Z³, Z⁴, Z⁵,Z⁶, and Z⁷ are each independently H, halo, alkyl, cycloalkyl,alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′,CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′,SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl,aryl and heteroaryl are optionally substituted; or one or more of Z¹ andZ², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷ together form anoptionally substituted carbocyclic or heterocyclic ring, and theremainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H,halo, alkyl, cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″,NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″,OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl,cycloalkyl, alkylene-aryl, aryl and heteroaryl are optionallysubstituted; X¹ and X² are each independently O, H or OR′; Y¹ is N orCR′; Y² is NR′, S, O or CR′; Y³ is N or CR′; W¹ and W² are both absentor are each independently H, OR′, NR′ or SR′; R¹ is H or an optionallysubstituted alkyl; and R′ and R″ are each independently H or anoptionally substituted alkyl or alkylene-aryl.
 3. The use of claim 1 or2, wherein the compound of Formula I is a compound of Formula I(a):

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷, are each independently H, halo,optionally substituted alkyl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″,CONR′R″, NR′COR″, OR′, SR′, CN, or CF₃; or Z⁶ and Z⁷ together form anoptionally substituted aromatic ring, and Z¹, Z², Z³, Z⁴, and Z⁵ areeach independently H, halo, optionally substituted alkyl, aryl,heteroaryl, NO₂, NR′R″, NR′SO₂R″, CONR′R″, NR′COR″, OR′, SR′, CN, orCF₃; R¹, R², R³, and R⁴ are each independently H or an optionallysubstituted alkyl; and R′ and R″ are each independently H, or anoptionally substituted alkyl.
 4. The use of any one of claims 1 to 3,wherein R¹ is C₁₋₆alkyl.
 5. The use of claim 4, wherein R¹ is methyl. 6.The use of any one of claims 3 to 5, wherein R² and R⁴ are both H. 7.The use of any one of claims 3 to 6, wherein R³ is H.
 8. The use of anyone of claims 1 to 7, wherein Z⁵ is OR′, wherein R′ is alkyl.
 9. The useof claim 8, wherein Z⁵ is OCH₃.
 10. The use of any one of claims 1 to 9,wherein Z¹, Z³ and Z⁴ are all H.
 11. The use of any one of claims 1 to10, wherein Z² is halo or H.
 12. The use of claim 11, wherein Z² ischloro.
 13. The use of any one of claims 1 to 12, wherein Z⁶ and Z⁷together form a substituted aromatic ring or unsubstituted aromaticring.
 14. The use of claim 13, wherein Z⁶ and Z⁷ together form anunsubstituted, 6-membered aromatic ring.
 15. The use of claim 1 or 2,wherein the compound is a compound of Formula III:


16. The use of claim 15, wherein the compound is cladoniamide A.
 17. Theuse of any one of claims 1 to 16, wherein the compound or thepharmaceutically acceptable salt thereof is the compound.
 18. The use ofany one of claims 1 to 17, wherein the viral infection is caused by oneor more coronavirus and/or flavivirus.
 19. The use of claim 18, whereinthe viral infection is caused by one or more coronavirus.
 20. The use ofclaim 19, wherein the viral infection is caused by one or more of severeacute respiratory syndrome (SARS) coronavirus-1 (SARS-CoV-1), SARScoronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome (MERS)coronavirus (MERS-CoV) and human coronavirus 229E (HCoV-229E).
 21. Theuse of claim 20, wherein the viral infection is caused by SARS-CoV-2.22. The use of claim 21, wherein the SARS-CoV-2 is SARS-CoV-2 SouthAfrica (501Y.V2), SARS-CoV-2 UK (VOC 202012/01) or SARS-CoV-2 Nigeria.23. The use of claim 20, wherein the viral infection is caused byHCoV-229E.
 24. The use of claim 18, wherein the viral infection iscaused by one or more flavivirus.
 25. The use of claim 24, wherein theviral infection is caused by one or more of dengue virus (DENV), WestNile virus (WNV), Zika virus (ZIKV), Powassan virus (POWV), Japaneseencephalitis virus (JEV) and yellow fever virus (YFV).
 26. The use ofclaim 25, wherein the viral infection is caused by one or more of WestNile virus (WNV), Powassan virus (POWV), Japanese encephalitis virus(JEV) and yellow fever virus (YFV).
 27. The use of claim 25, wherein theviral infection is caused by dengue virus (DENV).
 28. The use of claim25, wherein the viral infection is caused by Zika virus (ZIKV).
 29. Theuse of any one of claims 1 to 28, wherein the subject is a human.
 30. Acompound of Formula I(b):

wherein each

independently represents a single or double bond; Z¹, Z², Z³, Z⁴, Z⁵,Z⁶, and Z⁷ are each independently H, halo, alkyl, cycloalkyl,alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′,CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′,SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl,aryl and heteroaryl are optionally substituted; or one or more of Z¹ andZ², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷ together form anoptionally substituted carbocyclic or heterocyclic ring, and theremainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H,halo, alkyl, cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″,NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″,OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl,cycloalkyl, alkylene-aryl, aryl and heteroaryl are optionallysubstituted; X¹ and X² are each independently O, H or OR′; Y¹ is N orCR′; Y² is NR′, S, O or CR′; Y³ is N or CR′; W¹ and W² are both absentor are each independently H, OR′, NR′ or SR′; R¹ is H or an optionallysubstituted alkyl; and R′ and R″ are each independently H or anoptionally substituted alkyl or alkylene-aryl; or a pharmaceuticallyacceptable salt thereof, with the proviso that said compound is notexactly:

 or wherein R⁵ is C₁₋₄akyl, X² is selected from H and halo, and R¹ isselected from H, halo and OH.
 31. A use of a compound of Formula I or apharmaceutically acceptable salt thereof for inhibitingvacuolar-H⁺ATPase (V-ATPase):

wherein each

independently represents a single or double bond; Z¹, Z², Z³, Z⁴, Z⁵,Z⁶, and Z⁷ are each independently H, halo, alkyl, cycloalkyl,alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″, NR′SO₂R″, CO₂R′, COR′,CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″, OR′, SR′, SO₂R′, SOR′,SO₂NR′R″, CN or haloalkyl, wherein the alkyl, cycloalkyl, alkylene-aryl,aryl and heteroaryl are optionally substituted; or one or more of Z¹ andZ², Z² and Z³, Z³ and Z⁴, Z⁵ and Z⁶ and Z⁶ and Z⁷ together form anoptionally substituted carbocyclic or heterocyclic ring, and theremainder of Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H,halo, alkyl, cycloalkyl, alkylene-aryl, aryl, heteroaryl, NO₂, NR′R″,NR′SO₂R″, CO₂R′, COR′, CS₂R′, CSR′, CONR′R″, NR′COR″, CSNR′R″, NR′CSR″,OR′, SR′, SO₂R′, SOR′, SO₂NR′R″, CN or haloalkyl, wherein the alkyl,cycloalkyl, alkylene-aryl, aryl and heteroaryl are optionallysubstituted; X¹ and X² are each independently O, H or OR′; Y¹ is N orCR′; Y² is NR′, S, O or CR′; Y³ is N or CR′; W¹ and W² are both absentor are each independently H, OR′, NR′ or SR′; R¹ is H or an optionallysubstituted alkyl; and R′ and R″ are each independently H or anoptionally substituted alkyl or alkylene-aryl.
 32. The use of claim 31,wherein the compound or the pharmaceutically acceptable salt thereof isas defined in any one of claims 3 to
 17. 33. The use of claim 31 or 32,wherein the inhibition of V-ATPase is in a subject.
 34. The use of claim33, wherein the subject suffers from a viral infection.
 35. The use ofclaim 34, wherein the viral infection is as defined in any one of claims18 to
 28. 36. The use of any one of claims 31 to 35, wherein the subjectis a human.
 37. The use of any one of claims 1 to 29 and 31 to 36,wherein the compound is formulated for administration in apharmaceutical composition comprising the compound and optionally apharmaceutically acceptable carrier.